EP2383351A1

EP2383351A1 - A method for increasing the specificity of enzymatic dna synthesis

Info

Publication number: EP2383351A1
Application number: EP11173313A
Authority: EP
Inventors: Konstantin Ignatov; Vladimir Kramarov
Original assignee: Bioline Ltd
Current assignee: Bioline Ltd
Priority date: 2004-11-30
Filing date: 2005-11-25
Publication date: 2011-11-02
Also published as: GB0426303D0; EP1817428A1; GB2420560A; JP2011254833A; ATE544868T1; WO2006059075A1; EP1817428B1; JP2008521395A; JP4960250B2

Abstract

The invention provides a method of performing a DNA polymerase reaction by including in a reaction mixture containing a DNA polymerase a compound of the formula (I): wherein: R₁, R₂, R₃, R₄, R₅ may be the same or different and are independently selected from the group consisting of -, -OH, -F, -Cl, -Br, -CF₃ -CCl₃, - CBr₃, -NH₂, -NO₂, -CN, -CONH₂, -CHO, -CO₂H , -SH, -SO₃H, -OPO₃H₂ and a group R₇, R₆ is selected from the group consisting of -0-, -OPO₃H₂, -CF₃, -CCl₃, -CBr₃, -CN, -CONH₂, -CHO, -CO₂H, and a group R₇; each group R₇ is independently selected from C_1-6alkyl, C_2-6alkenyl, C_2-6alkynyl, or an aryl or heteroaryl ring containing from 5 to 8 ring members, which in the case of heteroaryl rings may include 1 or 2 nitrogen, oxygen or sulphur atoms or mixtures thereof, the remainder being carbon, and wherein R₇ is optionally be substituted by from 1 to 4 groups selected from -OH, -F, -Cl, -Br, -I, -CF₃ - CCl₃, -CBr₃, -NH-NH₂, -NH₂, -NO, -NO₂, -CN, -CONH₂, -CHO, -CO₂H , - SH, -SO₃H, -OPO₃H₂, or mixtures thereof, and further optionally R₇ may comprise a group -NH-NH-, -NN-, -NH-, -CO-, -S-S-. Kits comprising a DNA polymerase and compounds of the formula (I) also form part of the invention.

Description

Field of the Invention.

The present invention relates to improvements in methods of enzymatic synthesis of nucleic acids by the addition of organic compounds, such as 3-(1-pyridinio)-1-propanesulfonate (PPS), pyridine-N-oxide and others, containing a positive charged N-pyridinium group in molecules. In particular, the invention provides methods and kits for improvements in laboratory procedures using DNA polymerases. The disclosed methods are particularly useful for improving the specificity and yield of polymerase chain reaction (PCR).

Background to the Invention.

Polymerase chain reaction (PCR) is very important to the development of the biotechnology industry as well as for basic biological research. PCR reactions today are carried out by the use of a heat-resistant DNA polymerase enzyme (such as Taq DNA polymerase) in a multi-cycle process employing several alternating heating and cooling steps to amplify DNA ( U.S. Pat. Nos. 4,683,202 and 4,683,195 ). First, a reaction mixture is heated to a temperature sufficient to denature the double stranded target DNA into its two single strands. The temperature of the reaction mixture is then decreased to allow specific oligonucleotide primers to anneal to their respective complementary single-stranded target DNA. Following the annealing step, the temperature is raised to the temperature optimum of the DNA polymerase being used, which allows incorporation of complementary nucleotides at the 3' ends of the annealed oligonucleotide primers thereby recreating double stranded target DNA. Using a heat-stable DNA polymerase, the cycle of denaturing, annealing and extension may be repeated as many times as necessary to generate a desired product, without the addition of polymerase after each heat denaturation. Twenty or thirty replication cycles can yield up to a million-fold amplification of the target DNA sequence ("Current Protocols in Molecular Biology," F.M. Ausubel et al. (Eds.), John Wiley and Sons, Inc., 1998).
Although PCR technology has had a profound impact on biomedical research and genetic identity analysis, amplification of non-target oligonucleotides and mispriming on non-target background DNA, RNA, and/or the primers themselves, still present significant problems. This is especially true in diagnostic applications where PCR is carried out in a milieu of complex genetic backgrounds where the target DNA may be proportionately present at a very low level (Chou et al., Nucleic Acid Res., 20:1717-1723 (1992), and in PCR-amplifications of GC-rich DNA templates. A chief problem is that primers may prime extensions at non-specific sequences because only a few base pairs at the 3'-end of a primer, which are complementary to a DNA sequence, can result in a stable priming complex. As a result, competitive or inhibitory products can be produced at the expense of the desired product.
The numerous protocols designed for different PCR applications almost always require an optimization step for implementation (Dieffenbach,C.W. and Dveksler,G.S. (1995) PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, NY.). In some cases, especially when complex cDNA or genomic DNA is used as the PCR template and the calculated annealing temperature is relatively low, multiple non-specific bands cannot be eliminated although all experimental parameters are seemingly optimized. It has been reported that some reagents like tetramethylammonium (TMA) chloride (Chevet, E., et al, (1995) Nucleic Acids Res., 23, 3343-3344; Hung, T., et al, (1990) Nucleic Acids Res., 18, 4953; Warner, C.K. and Dawson, J.E. (1996) In Persing, D.H. (ed.), PCR Protocols for Emerging Infectious Diseases. ASM Press, Washington DC.), dimethyl sulfoxide (Winship, P.R. (1989) Nucleic Acids Res., 17, 1266; Bookstein, R., et al, (1990) Nucleic Acids Res., 18, 1666; Sidhu, M.K., et al, (1996) Biotechniques, 21, 44-47.), amides (Chakrabarti, R., et al, (2001) Nucleic Acids Res., 29, 2377-2381) and betaine (Weissensteiner, T., et al, (1996) Biotechniques, 21, 1102-1108; Henke, W., et al, (1997) Nucleic Acids Res., 19, 3957; U.S. Pat. Nos. 6,270,962 ) are capable of improving the efficacy and specificity of PCR. These reagents are often used as components of the commercial optimization and enhancer kits for PCR.
Unfortunately, in a number of cases, the above mentioned compounds are unable to suppress non-specific PCR products or increase the yield of specific fragments. Thus, there is a need for new compounds for use as PCR enhancers.

Disclosure of the Invention.

The present invention provides a method for improvement of DNA enzymatic synthesis. In particular, the invention relates primarily to the improvement of PCR specificity and yield by the addition of organic compounds containing a positive charged pyridinium group in their molecules, such as N-alkylpyridinium salts (e.g. 1-propylpyridinium bromide), pyridinium inner salts (e.g. 3-(1-pyridinio)-1-propanesulfonate), pyridine-N-oxide and others. The invention also can be useful for improving other laboratory procedures using DNA polymerases, such as primer extension and DNA sequencing.
The present invention provides methods, regents and reaction kits for suppressing the non-specific products and/or increasing the yield of specific products in reactions catalyzed by DNA polymerase. The decrease in non-specific products and the increase in specific products of the reactions are achieved by the addition of organic compounds containing a positive charged N-pyridinium group in molecules, which are described herein, to the reaction mixtures.
In particular, the invention provides a method of performing a DNA polymerase reaction by including in a reaction mixture containing a DNA polymerase a compound of the formula I:
wherein:

R₁, R₂, R₃, R₄, R₅ may be the same or different and are independently selected from the group consisting of -H, -OH, -F, - Cl, -Br, -CF₃, -CCl₃, -CBr₃, -NH₂, -NO₂, -CN, -CONH₂, -CHO, -CO₂H, - SH, -SO₃H, -OPO₃H₂ and a group R₇,
R₆ is selected from the group consisting of -O^- -OPO₃H₂, -CF₃, -CCl₃, -CBr₃, -CN, -CONH₂, -CHO, -CO₂H and a group R₇;
each group R₇ is independently selected from C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or an aryl or heteroaryl ring containing from 5 to 8 ring members, which in the case of heteroaryl rings may include 1 or 2 nitrogen, oxygen or sulphur atoms or mixtures thereof, the remainder being carbon, and wherein R₇ is optionally be substituted by from 1 to 4 groups selected from -OH, -F, -Cl, - Br, -I, -CF₃, -CCl₃, -CBr₃, -NH-NH₂, , -NH₂, -NO, -NO₂, -CN, -CONH₂, -CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂, or mixtures thereof, and further optionally R₇ may comprise a group -NH-NH-, -NN-, -NH-, -CO-, -S-S-.

The present invention allows improving the efficacy and specificity of DNA polymerase reactions, such as primer extension reaction, reverse-transcription reaction, DNA sequencing, nick-translation, PCR and other reactions which are catalyzed by DNA polymerases.
The organic compounds containing a positive charged N-pyridinium group in molecules, which are capable of improving the efficacy and specificity of DNA polymerase reactions, may be N-alkylpyridinium salts (e.g. 1-propylpyridinium bromide), pyridinium inner salts (e.g. 3-(1-pyridinio)-1-propanesulfonate), pyridine-N-oxide and others described herein.
The DNA polymerases, which are used for performing the DNA polymerase reactions, can be RNA-dependent DNA polymerases, such as AMV or MMLV reverse-transcriptase, and DNA-dependent DNA polymerases, such as Klenow fragment, DNA Polymerase I E.coli, Taq, Tth or Pfu DNA polymerase.
The reaction kits of the invention contain the organic compounds, which are necessary to improve the specificity and/or efficacy of DNA polymerase reactions and described herein. Furthermore, the reaction kit may contain a plurality of additional reaction components. Among the additional reaction components, one may include an enzyme, such as a DNA polymerase for PCR, reverse-transcription or DNA sequencing.
Other features, aspects and advantages of the invention will be, or will become, apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, features, aspects and advantages included within this description, are within the scope of the invention, and are protected by the following claims.

Brief Description of the Figures.

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, claims and accompanying drawings where:

FIG. 1 depicts an electrophoretic analysis of the PCR products obtained in Example 1. A 614-bp DNA fragment was amplified from 25 ng of Gallus domesticus genomic DNA for 30 cycles. PCR was performed under conventional conditions without extra additives (lane 1), in presence of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M pyridine-N-oxide ( lanes 2, 3, 4, 5, 6), and in presence of 0.1 M, 0.2 M, 0.3 M, 0.4 M 3-(1-pyridinio)-1-propanesulfonate ( lanes 7, 8, 9, 10).
FIG. 2 depicts an electrophoretic analysis of the PCR products obtained in Example 2. A 614-bp DNA fragment was amplified from 25 ng of Gallus domesticus genomic DNA for 30 cycles. PCR was performed under conventional conditions without extra additives (lane 1); in presence of 0.2 M and 0.4 M 3-(1-pyridinio)-1-propanesulfonate (lanes 2 and 3); in presence of 0.3 M, 0.6 M and 1.2 M betaine ( lanes 4, 5, and 6); in presence of 12.5 mM, 25 mM and 50mM tetramethylammonium chloride (TMA-Cl) ( lanes 7, 8, and 9); and in presence of 0.7 M, 1.4 M and 2.1 M DMSO ( lanes 10, 11, and 12).
FIG. 3 depicts an electrophoretic analysis of the PCR products obtained in Example 3. A 1450-bp DNA fragment of X. laevis cDNA was amplified from 100 ng of Xenopus laevis embryo MATCHMAKER LexA cDNA library (Clontech Laboratories) in 40 cycles. PCR was performed under conventional conditions without extra additives (lane 1); in presence of 1.25 M and 1.7M betaine (lanes 2 and 3); and in presence of 0.3 M, 0.35 M and 0.4 M 3-(1-pyridinio)-1-propanesulfonate ( lanes 4, 5 and 6). Lane M - DNA marker.
FIG. 4 depicts an electrophoretic analysis of the PCR products obtained in Example 4. A 614-bp DNA fragment was amplified from 25 ng of Gallus domesticus genomic DNA for 30 cycles. PCR was performed under conventional conditions without extra additives (lane 1); in presence of 0.1 M, 0.2 M, 0.3 M, 0.4 M and 0.5M pyridine-N-oxide ( lanes 2, 3, 4, 5 and 6); and in presence of 0.1 M, 0.2 M, 0.3 M, 0.4 M and 0.5M trimethylamine-N-oxide ( lanes 7, 8, 9, 10 and 11).

Detailed Description of the Invention.

In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided.
The term "nucleic acids", as used herein, refers to either DNA or RNA. It includes plasmids, chromosomal DNA or RNA, nonfunctional DNA or RNA, and DNA or RNA synthesized in vitro.
The term "nucleic acid sequence" or "polynucleotide sequence" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
The terms "oligonucleotide primer", "oligonucleotide" or "primer" refer to a single-stranded polymer of deoxyribonucleotides or ribonucleotides.
The term "complementary" as used herein refers to a relationship between two nucleic acid sequences. One nucleic acid sequence is complementary to a second nucleic acid sequence if it is capable of forming a duplex with the second nucleic acid, wherein each residue of the duplex forms a guanosine-cytidine (G-C) or adenosine-thymidine (A-T) base pair or an equivalent base pair. Equivalent base pairs can include nucleoside or nucleotide analogues other than guanosine, cytidine, adenosine, or thymidine.
Terms "thermostable", "thermally stable" and "heat-stable" are used interchangeably herein to describe enzymes, which can withstand temperatures up to at least 95°C for several minutes without becoming irreversibly denatured. Typically, such enzymes have an optimum temperature above 45°C, preferably between 50° to 75°C.
The term "modification" of an enzyme as used herein, refers to a chemical or genetic modification of enzyme.
The terms "DNA template", "RNA template" or "template" as used herein, refer to a nucleic acid that is used by a polymerase to synthesize a new complementary nucleic acid.
The term "betaine", as used herein, refers to N,N,N-trimethylglycine.
The term "pyridinium betain", as used herein, refers to inner salts of pyridinium.
Some compounds of the invention may be present with a positive or negative charge or with both a positive and negative charges, depending on the pH of the solution. It is understood that these various forms of these compounds are included in the present invention.
In one aspect, the present invention provides reagents and methods for suppressing the non-specific products and increasing the yield of specific products in DNA polymerase reactions. These reactions can be a primer extension reaction, a reverse-transcription reaction, DNA sequencing, nick-translation, PCR and other reactions, which are catalyzed by DNA polymerases. Suppressing the non-specific products and increasing the yield of specific products of the reactions are achieved by the addition of organic compounds containing a positive charged pyridinium group. These compounds, which are capable of improving the efficacy and specificity of DNA polymerase reactions, can be N-alkylpyridinium salts (e.g. 1-propylpyridinium bromide), pyridinium inner salts (e.g. 3-(1-pyridinio)-1-propanesulfonate), pyridine-N-oxide and others.
In one aspect, the present invention provides a reaction kit for increasing the specificity and/or efficacy of DNA polymerase reactions. The kit may include an organic compound (or compounds) containing a positive charged pyridinium group in its molecules, or a complex reagent containing this compound, or solution of the compound or the reagent. The kit may further include one or more additional reaction components to facilitate the enzymatic process. The kit may further include one or more DNA polymerases for performing the enzymatic process.
Generally, a kit may comprise a first container containing a compound of the formula I and at least a second container having one or more components suitable for performing a DNA polymerase reaction. The second container may contain one of more of (a) dNTPs; (b) ddNTPs; (c) a DNA polymerase; (d) reaction buffer(s) and (e) a primer. The kit may contain two or more, e.g. three, four or five separate containers with these or other components packaged separately or in combinations thereof. Kits may also contain instructions for use of the reagents.
The kit will usually contain the compound of formula I in a concentrated solution, e.g. at about 5x to 10x the reaction concentration.
Suitable DNA polymerases for use in the methods, kits and other aspects of the invention include E. coli DNA Polymerase I, Klenow enzyme, T4 DNA Polymerase, and T7 DNA Polymerase. These polymerases may be of wild-type sequences or synthetic variants and fragments.
The polymerase may be a reverse transcriptase, such as AMV or MMLV reverse-transcriptase, including synthetic variants or fragments thereof.
In a preferred aspect, the DNA polymerase will be a thermally stable DNA polymerase. This includes, but is not limited to, Thermus aquaticus DNA polymerase; N-terminal deletions of Taq polymerase, including the Stoffel fragment of DNA polymerase, Klentaq-235, and Klentaq-278; Thermus thermophilus DNA polymerase; Bacillus caldotenax DNA polymerase; Thermus flavus DNA polymerase; Bacillus stearothermophilus DNA polymerase; and archaebacterial DNA polymerases, such as Thermococcus litoralis DNA polymerase (also referred to as Vent_R ^®), Pfu, Pfx, Pwo, and DeepVent_R ^® or a mixture thereof. Other commercially available polymerases DNA polymerases include TaqLA or Expand High Fidelity^plus Enzyme Blend (Roche); KlenTaqLA, KlenTaq1, TthLA (Perkin-Elmer), ExTaq® (Takara Shuzo); Elongase® (Life Technologies); Taquenase™ (Amersham), TthXL (Perkin Elmer); Advantage™ KlenTaq and Advantage™ Tth (Clontech); TaqPlus® and TaqExtender™ (Stratagene); or mixtures thereof.
Methods for using compounds containing a positive charged pyridinium group for increasing the specificity and/or efficacy of DNA polymerase reactions are also provided in which the compound is added to a reaction mixture. Additional steps include incorporation of additional reaction components, which may include template nucleic acid(s), oligonucleotide primer(s), polymerase(s) and others, and adjusting the reaction conditions (e.g., increased temperature) to activate and/or complete an enzymatic process.
In a preferred embodiment, the present invention includes reagents and methods for increasing the specificity of PCR. Specifically, the present invention provides processes and kits for performing a specific PCR. The processes and kits utilize the step of addition of the organic compounds, which are capable of improving the PCR specificity and described herein, to the reaction mixture of PCR.
PCR is a polymerase chain reaction described in US Patents Nos. 4,683,202 and 4,683,195 . The PCR employs a heating and cooling cycle to drive the reaction. First, the reaction mixture is heated to, or above, the nucleic acid melting temperature, then cooled to allow specific oligonucleotide primers to bind to the sample (annealing), and then heated to optimize the addition of complementary bases to the amplified nucleic acid (extension). The use of a heat-stable DNA polymerase ( U.S. Pat. No. 4,889,818 ) allows to repeat this cycle of denaturing, annealing and extension as many times as needed to generate the desired product. Twenty replication cycles can yield up to a million-fold amplification of the target DNA sequence.
Preferred DNA polymerases for use in PCR applications include thermally stable DNA polymerases and/or combinations thereof. Thermally stable DNA polymerases may include, but are not limited those mentioned herein above.
The addition of an appropriate amount of one or more of the compounds described herein facilitates the improving the efficacy and specificity of PCR. See examples herein, for a demonstration of the effects of 1-propylpyridinium bromide, 3-(1-pyridinio)-1-propanesulfonate and pyridine-N-oxide on PCR efficacy and specificity. Other compounds of the invention can be used in a similar manner to improve efficacy and specificity of PCR methods
As indicated above, the compounds used in the present invention are N-pyridinium group containing organic molecules or ions that are capable of eliminating or reducing the non-specific products of PCR and other laboratory procedures using DNA polymerases, such as reverse-transcription or DNA sequencing. These compounds are represented by the formula I defined herein above.
In one embodiment, in the compounds of formula I the groups R₁, R₂, R₃, R₄, and R₅ may be the same or different and are independently selected from the group consisting of -H, -CO₂H, -OH, -Cl, -Br, - NH₂ or C_1-6 alkyl optionally substituted by a group -SO₃H, -CO₂H, - OPO₃H₂ or -OH, and, R₆ may be selected from the group consisting of -O-, -OPO₃H₂, -CO₂H, -CONH₂, -CHO or C_1-6alkyl optionally substituted by a group -SO₃H, -CO₂H, -OPO₃H₂ or -OH.
In a preferred embodiment, the groups R₁, R₂, R₄, and R₅ are hydrogen, the group R₃ is hydrogen or a C_1-6, alkyl optionally substituted by a group -SO₃ ^-, -CO₂ ^- or -OH, and the group R₆ is as defined above for formula I.
In another embodiment, the groups R₁, R₂, R₄, and R₅ are hydrogen, the group R₃ is an unbranched C_1-4 alkyl optionally substituted at its terminal carbon by a group a group -SO₃ ^-, -CO₂ ^- or -OH, and the group R₆ is as defined above for formula I.
Preferred groups R₆ include linear or branched C_1-6 alkyl groups, optionally substituted by a group -SO₃ ^-; -CO₂ ^-; -OH; oxygen; or a 4-pyridyl group or acid addition salt thereof. The C_1-6 alkyl groups are most preferably methyl, ethyl, n-propyl, n-butyl, n-amyl or n-hexyl optionally substituted by a group -SO₃ ^-, -CO₂ ^- or -OH. It will be understood that the preferred groups R₆ are also preferred in conjunction with the preferred groups R₁, R₂, R₃, R₄, R₅ described in the preceding two paragraphs.
Some compounds, which are represented by formula I, may be present with a positive or with both a positive and negative charges, depending on the pH of the solution. It is understood that these various forms of these compounds are included in the present invention.
It will thus be appreciated that groups such as -CONH₂, -CO₂H, - SO₃H, -OPO₃H₂ in particular may exist as their corresponding charged radicals and reference herein to such groups will be understood to include their charged forms, as well as salts thereof.

Preferred compounds for improving PCR applications can be selected from, but are not limited by, the group compounds set out in table 1:

Compound	R1	R2	R3	R4	R5	R6
2-(1-pyridino)-1-ethanesulfonate; or 1-(2-Sulfoethyl)pyridinium betain	H	H	H	H	H	-(CH₂)₂-SO₃H
3-(1-pyridino)-1-propanesulfonate; or 1-(3-Sulfopropyl)pyridinium betain	H	H	H	H	H	-(CH₂)₃-SO₃H
1-methyl-4-(3-sulfopropyl)pyridinium betain	H	H	(CH₂)₃-SO3-	H	H	Methyl
1-methylpyridinium chloride	H	H	H	H	H	Methyl
1-methylpyridinium bromide	H	H	H	H	H	Methyl
1-ethylpyridinium chloride	H	H	H	H	H	Ethyl
1-ethylpyridinium bromide	H	H	H	H	H	Ethyl
1-propylpyridinium chloride	H	H	H	H	H	n-Propyl
1-Propylpyridinium bromide	H	H	H	H	H	n-Propyl
1-butylpyridinium chloride	H	H	H	H	H	n-Butyl
1-butylpyridinium bromide	H	H	H	H	H	n-Butyl
1-Butyl-4-methylpyridinium chloride	H	H	Methyl	H	H	n-Butyl
1-Butyl-4-methylpyridinium chloride	H	H	Methyl	H	H	n-Butyl
pyridine-N-oxide	H	H	H	H	H	-O^-
2-Hydroxypyridine N-oxide	H	H	H	H	H	-O^-
1-(4-pyridyl)pyridinium chloride hydrochloride	H	H	H	H	H	-4-pyridyl-hydrochloride

In general, the compounds used to improve PCR, DNA sequencing and other laboratory procedures using a DNA polymerase are commercially available. For example, 3-(1-pyridino)-1-propanesulfonat, 1-butyl-4-methylpyridinium chloride, pyridine-N-oxide, 2-hydroxypyridine-N-oxide (2-pyridinol 1-oxide) and 1-(4-pyridyl)pyridinium chloride hydrochloride can all be obtained from Fluka, 1-propylpyridinium bromide can be obtained from Sigma. These compounds may also be synthesized by routine methods known to those of skill in the art.
In a preferred embodiment, the present invention includes methods for increasing the specificity of PCR. Specifically, the present invention provides processes, reagents and kits for suppressing the non-specific products and/or increasing the yield of specific PCR products. The processes, reagents and kits utilize the organic compounds containing a positive charged N-pyridinium group in molecules. By using methods of the present invention, the PCR-amplification specificity of the target DNA molecules is increased.
The present procedures and reagents can be applied to PCR processes as set forth in Examples.
The principles, methodologies and examples described herein (and below) for performing PCR may be applied in an analogous fashion to perform various types of DNA polymerase reactions described above.
To perform a DNA polymerase reaction according to the present invention, a reaction mixture which includes a DNA polymerase is provided, together with a suitable concentration of a compound of formula I. Typically, the compound of formula I (or mixture of such compounds) may be provided at a final concentration of from 0.05 to 1.0 M, preferably from 0.1 to 0.9 M, such as from 0.2 to 0.75 M, more preferably from 0.2 to 0.5 M, e.g. from 0.25 to 0.5 M.
In addition to the DNA polymerase the reaction mixture typically contains other components conventional in the art such as a DNA template, one or more primers, dNTPs and a buffer for the polymerase. These may be provided by a person of skill in the art using routine skill and knowledge and the precise nature and amounts of these components is not essential as such to the present invention.
The following Examples illustrate aspects of the invention. The Examples are illustrative of, but not binding on, the present invention. Any methods, preparations, solutions and such like, which are not specifically defined, may be found in Sambrook et al. All solutions are aqueous and made up in sterile, deionised water, unless otherwise specified. All enzymes were obtained from Promega Corporation.

EXAMPLES

Example 1

The improvement of PCR specificity and yield by adding compounds containing a positively charged pyridinium group in molecules (pyridine-N-oxide and 3-(1-pyridinio)-1-propanesulfonate).
A 614-bp DNA fragment was amplified from 25 ng of Gallus domesticus genomic DNA in 30 cycles: 95°C - 30 sec; 58°C - 30 sec; 72°C - 30 sec. The reaction mixture (50 µl) contained: 1.5 mM MgCl₂, 20 mM Tris-HCl (pH 9.0 at 25°C.), 50 mM KCl, 0.1% Triton X-100, 0.2 mM each dNTP, 20 pmol primer Pr1 (5'-attactcgagatcctggacaccagc), 20 pmol primer Pr2 (5'-attaggatcctgccctctcccca), and 2.5U Taq DNA polymerase.
PCR reactions were performed in the absence of any extra additives and in the presence of extra additives: pyridine-N-oxide or 3-(1-pyridinio)-1-propanesulfonate. The extra additives were added to the reaction mixture up to final concentrations: 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M pyridine-N-oxide, and 0.1 M, 0.2 M, 0.3 M, 0.4 M 3-(1-pyridinio)-1-propanesulfonate.
FIG. 1 depicts the electrophoretic analysis of the amplification products obtained. Under these reaction conditions only the method of present invention (addition of the compounds containing a positive charged pyridinium group in molecules) provided a detectable amount of the desired product (increased yield of PCR) and elimination of non-specific amplification products (increased specificity of PCR). Compared to the conventional PCR procedure without extra additives (lane 1), target DNA synthesis and elimination of non-specific PCR products were obtained by using pyridine-N-oxide at concentrations from 0.3 M to 0.5 M (lanes 4-6) or 3-(1-pyridinio)-1-propanesulfonate at concentrations from 0.2 M to 0.4 M (lanes 8-10) (note the presence of the target amplification product and the absence of non-specific products in lanes 4-6 and lanes 8-10 compared to lane 1).

Example 2

Comparing the ability of betaine, tetramethylammonium chloride (TMA-Cl), dimethyl sulfoxide (DMSO) and 3-(1-pyridinio)-1-propanesulfonate to improve the specificity of PCR.
Betaine, tetramethylammonium chloride (TMA-Cl) and dimethyl sulfoxide (DMSO) are the most popular additives to DNA polymerase reaction mixtures (e.g. PCR and DNA Sequencing mixtures), which are used for improving specificity and yield of the reactions. The ability of betaine, TMA-Cl, DMSO and claimed in this invention 3-(1-pyridinio)-1-propanesulfonate to improve specificity and yield of a DNA polymerase reaction were compared in PCR-amplification of 614-bp DNA fragment from Gallus domesticus genomic DNA.
PCR was performed as described in the Example 1. The reaction mixtures contained or did not contain the extra additives.
FIG. 2 depicts the electrophoretic analysis of the amplification products obtained. Under these reaction conditions only the presence of 0.2 - 0.4 M 3-(1-pyridinio)-1-propanesulfonate (lanes 2 and 3) and betaine at concentrations higher than 1 M (note lane 6 - 1.2 M betaine) provided an elimination of non-specific amplification products along with a detectable amount of the desired product.
It should be noted that 0.2 M 3-(1-pyridinio)-1-propanesulfonate provides the same effect as 1.2 M betaine. An additional comparison between betaine and 3-(1-pyridinio)-1-propanesulfonate is described in Example 3.

Example 3

Comparing the ability of betaine (claimed in U.S. Pat. Nos. 6,270,962 ) and 3-(1-pyridinio)-1-propanesulfonate (claimed in this invention) to improve PCR-amplification of DNA.
A 1450-bp DNA fragment of X. laevis cDNA was amplified from 100 ng of Xenopus laevis embryo MATCHMAKER LexA cDNA library (Clontech Laboratories) in 40 cycles: 94°C - 30 sec; 58°C - 35 sec; 72°C - 60 sec. The reaction mixture (50 µl) contained: 1.5 mM MgCl₂, 20 mM Tris-HCl (pH 9.0 at 25°C.), 50 mM KCl, 0.1% Triton X-100, 0.2 mM each dNTP, 20 pmol primer Pr3 (5'-attaccatggacccagcggctcctgccacc), 20 pmol primer Pr4 (5'-ccgctcgagagctcctctacctccttc), and 2.5U Taq DNA polymerase.
PCR reactions were performed in the absence of any extra additives and in the presence of the following extra additives: 1.25 M or 1.7 M betaine, or 0.3 M, 0.35 M or 0.4 M 3-(1-pyridinio)-1-propanesulfonate.
FIG. 3 depicts the electrophoretic analysis of the amplification products obtained. Under these reaction conditions only the presence of 3-(1-pyridinio)-1-propanesulfonate (lanes 4-6) provided a detectable amount of the desired product. Compared to the conventional PCR procedure without extra additives (lane 1), the additions of both betaine (lanes 2 and 3) and 3-(1-pyridinio)-1-propanesulfonate (lanes 4-6) to the PCR mixture provided a decrease of non-specific amplification products, however, complete elimination of non-specific products was achieved only by 0.4 M 3-(1-pyridinio)-1-propanesulfonate (lane 6).

Example 4

Comparing the ability of trimethylamine-N-oxide (claimed in U.S. Pat. Nos. 6,270,962 ) and pyridine-N-oxide (claimed in this invention) to increase PCR specificity and yield.
Two N-oxide compounds, trimethylamine-N-oxide and pyridine-N-oxide, were tested as compounds increasing specificity and yield of a DNA polymerase reaction, such as PCR. Trimethylamine-N-oxide was described in U.S. Pat. Nos. 6,270,962 as a compound improving a DNA polymerase reaction, such as DNA sequencing. Claimed herein pyridine-N-oxide contains a positive charged pyridinium group instead of a positive charged N,N,N-trimethyl group in trimethylamine-N-oxide.
The PCR was performed as described in the Example 1. The reaction mixtures contained from 0.1 M to 0.5 M pyridine-N-oxide, or from 0.1 M to 0.5 M trimethylamine-N-oxide, or did not contain the extra additives.
FIG. 4 depicts the electrophoretic analysis of the amplification products obtained. Under these reaction conditions only the presence of 0.3 - 0.5 M pyridine-N-oxide (lanes 4-6) provided elimination of non-specific amplification products along with obtaining a detectable amount of the desired product. Compared to the conventional PCR procedure without extra additives (lane 1), trimethylamine-N-oxide at concentrations from 0.1 M to 0.5 M (lanes 7-11) did not provide a considerable improvement of PCR.
It is to be understood that the above-described methods are merely representative embodiments illustrating the principles of this invention and that other variations in the methods may be devised by those skilled in the art without departing from the spirit and scope of this invention.
The following numbered paragraphs contain statements of broad combinations of the inventive technical features herein disclosed:-

1. A method of performing a DNA polymerase reaction by including in a reaction mixture containing a DNA polymerase a compound of the formula I:
wherein:
- R₁, R₂, R₃, R₄, R₅ may be the same or different and are independently selected from the group consisting of -H, -OH, -F, - Cl, -Br, -CF₃, -CCl₃, -CBr₃, -NH₂, -NO₂, -CN, -CONH₂, -CHO, -CO₂H, - SH, -SO₃H, -OPO₃H₂ and a group R₇,
- R₆ is selected from the group consisting of -O^-, -OPO₃H₂, -CF₃, -CCl₃, -CBr₃, -CN, -CONH₂, -CHO, -CO₂H, and a group R₇;
- each group R₇ is independently selected from C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or an aryl or heteroaryl ring containing from 5 to 8 ring members, which in the case of heteroaryl rings may include 1 or 2 nitrogen, oxygen or sulphur atoms or mixtures thereof, the remainder being carbon, and wherein R₇ is optionally be substituted by from 1 to 4 groups selected from -OH, -F, -Cl, - Br, -I, -CF₃, -CCl₃, -CBr₃, -NH-NH₂, , -NH₂, -NO, -NO₂, -CN, -CONH₂, - CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂, or mixtures thereof, and further optionally R₇ may comprise a group -NH-NH-, -NN-, -NH-, -CO-, -S-S-.
2. A method according to paragraph 1 wherein the groups R₁, R₂, R₃, R₄, and R₅ are the same or different and are independently selected from the group consisting of -H, -CO₂H, -OH, -Cl, -Br, -NH₂ or C_1-6 alkyl optionally substituted by a group -SO₃H, -CO₂H, -OPO₃H₂ or -OH, and, R₆ is selected from the group consisting of -O-, -OPO₃H₂, -CO₂H, -CONH₂, -CHO or C_1-6alkyl optionally substituted by a group -SO₃H, - CO₂H, -OPO₃H₂ or -OH.
3. A method according to paragraph 1, wherein the groups R₁, R₂, R₃, R₄, R₅ are hydrogen and the group R₆ is as defined in paragraph 1.
4. A method according to paragraph 1, wherein the groups R₁, R₂, R₄, and R₅ are hydrogen, the group R₃ is a C_1-6, alkyl optionally substituted by a group -SO₃H, -CO₂H or -OH and the group R₆ is as defined in paragraph 1.
5. A method according to paragraph 1, wherein the groups R₁, R₂, R₄, and R₅ are hydrogen, the group R₃ is an unbranched C_1-4 alkyl optionally at its terminal carbon by a group -SO₃H, -CO₂H or -OH and the group R₆ is as defined in paragraph 1.
6. The method according to any one of paragraphs 2 to 5 wherein R₆ is a linear or branched C_1-6 alkyl group optionally substituted by a group -SO₃H, -CO₂H, -OH; oxygen; or a 4-pyridyl group or acid addition salt thereof.
7. The method according to paragraph 6 wherein R₆ is a methyl, ethyl, n-propyl, n-butyl, n-amyl or n-hexyl group optionally substituted by a group -SO₃ ^-, -CO₂ ^- or -OH.
8. The method of paragraph 6 wherein R₁, R₂, R₃, R₄, R₅ are hydrogen and R₆ is sulfomethyl, sulfoethyl, sulfopropyl or sulfobutyl.
9. The method according to paragraph 1 wherein said compound is pyridine-N-oxide.
10. The method according to paragraph 1 wherein said compound is 3-(1-pyridinio)-1-propanesulfonate.
11. The method according to paragraph 1 wherein said compound is 2-pyridinol 1-oxide.
12. The method according to any one of the preceding paragraphs wherein said DNA polymerase reaction is a PCR.
13. The method according to any one of paragraphs 1 to 11 wherein said DNA polymerase reaction is a reaction of chain termination DNA sequencing.
14. The method according to any one of paragraphs 1 to 11 wherein said DNA polymerase reaction is a primer extension reaction.
15. The method according to any one of paragraphs 1 to 11 wherein said DNA polymerase reaction is a reverse-transcription reaction.
16. The method according to any one of paragraphs 1 to 11 wherein said DNA polymerase reaction is a nick-translation reaction.
17. The method according to any one of the preceding paragraphs wherein the reaction mixture comprises a DNA molecule having trinucleotide repeats where the trinucleotides are GC rich.
18. A kit for performing a DNA polymerase reaction comprising in separate containers:
1. a) components for DNA polymerase reaction; and
2. b) a container which contains one or more compounds of the formula I:
  wherein:
  - R₁, R₂, R₃, R₄, R₅ may be the same or different and are independently selected from the group consisting of -H, -OH, -F, - Cl, -Br, -CF₃, -CCl₃, -CBr₃, -NH₂, -NO₂, -CN, -CONH₂, -CHO, -CO₂H, - SH, -SO₃H, -OPO₃H₂ and a group R₇,
  - R₆ is selected from the group consisting of -O^-, -OPO₃H₂, -CF₃, -CCl₃, -CBr₃, -CN, -CONH₂, -CHO, -CO₂H, and a group R₇,
  - each group R₇ is independently selected from C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or an aryl or heteroaryl ring containing from 5 to 8 ring members, which in the case of heteroaryl rings may include 1 or 2 nitrogen, oxygen or sulphur atoms or mixtures thereof, the remainder being carbon, and wherein R₇ is optionally be substituted by from 1 to 4 groups selected from -OH, -F, -Cl, - Br, -I, -CF₃, -CCl₃, -CBr₃, -NH-NH₂, , -NH₂, -NO, -NO₂, -CN, -CONH₂, - CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂, or mixtures thereof, and further optionally R₇ may comprise a group -NH-NH-, -NN-, -NH-, -CO-, -S-S-.
19. The kit of paragraph 18 wherein the compound of formula I is as defined in any one of paragraphs 2 to 11.
20. The kit of paragraph 18 or 19 further comprising a DNA polymerase enzyme.
21. The kit of paragraph 20 wherein said DNA polymerase enzyme is selected from the group consisting of E. coli DNA Polymerase I, Klenow enzyme, T4 DNA Polymerase, and T7 DNA Polymerase.
22. The kit of paragraph 20 wherein said DNA polymerase is a reverse transcriptase.
23. The kit of paragraph 22 wherein said reverse transcriptase is selected from the group consisting of AMV and M-MuLV.
24. The kit of paragraph 20 wherein said DNA polymerase is a thermostable DNA polymerase.
25. The kit of paragraph 24 wherein said thermostable DNA polymerase is selected from the group consisting of Taq DNA polymerase; N-terminal deletions of Taq polymerase, Tth DNA polymerase, Bacillus caldotenax DNA polymerase; Thermus flavus DNA polymerase; Bacillus stearothermophilus DNA polymerase; and archaebacterial DNA polymerases, such as Pfu DNA polymerase, Pfx DNA polymerase, Pwo DNA polymerase, Thermococcus litoralis DNA polymerase, and DeepVent_R ^®.
26. In a method of performing a DNA polymerase reaction, the improvement comprising including in a reaction mixture containing a DNA polymerase a compound of the formula I:
wherein:
- R₁, R₂, R₃, R₄, R₅ may be the same or different and are independently selected from the group consisting of -H, -OH, -F, - Cl, -Br, -CF₃, -CCl₃, -CBr₃, -NH₂, -NO₂, -CN, -CONH₂, -CHO, -CO₂H, - SH, -SO₃H, -OPO₃H₂ and a group R₇,
- R₆ is selected from the group consisting of -O^-, -OPO₃H₂, -CF₃, -CCl₃, -CBr₃, -CN, -CONH₂, -CHO, -CO₂H, and a group R₇,
- each group R₇ is independently selected from C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or an aryl or heteroaryl ring containing from 5 to 8 ring members, which in the case of heteroaryl rings may include 1 or 2 nitrogen, oxygen or sulphur atoms or mixtures thereof, the remainder being carbon, and wherein R₇ is optionally be substituted by from 1 to 4 groups selected from -OH, -F, -Cl, - Br, -I, -CF₃, -CCl₃, -CBr₃, -NH-NH₂, , -NH₂, -NO, -NO₂, -CN, -CONH₂, - CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂, or mixtures thereof, and further optionally R₇ may comprise a group -NH-NH-, -NN-, -NH-, -CO-, -S-S-.

Claims

A method of performing a DNA-dependent DNA polymerase reaction by including in a reaction mixture containing a DNA-dependent DNA polymerase a compound of the formula I:
wherein:
R₁, R₂, R₃, R₄, R₅ may be the same or different and are independently selected from the group consisting of -H, -OH, -F, -Cl, -Br, -CF₃, -CCl₃, -CBr₃, -NH₂, -NO₂, -CN, -CONH₂, -CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂ and a group R₇,

R₆ is selected from the group consisting of -O-, -OPO₃H₂, - CF₃, -CCl₃, -CBr₃, -CN, -CONH₂, -CHO, -CO₂H, and a group R₇;

each group R₇ is independently selected from C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or an aryl or heteroaryl ring containing from 5 to 8 ring members, which in the case of heteroaryl rings may include 1 or 2 nitrogen, oxygen or sulphur atoms or mixtures thereof, the remainder being carbon, and wherein R₇ is optionally be substituted by from 1 to 4 groups selected from - OH, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -NH-NH₂, , -NH₂, -NO, - NO₂, -CN, -CONH₂, -CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂, or mixtures thereof, and further optionally R₇ may comprise a group -NH-NH-, -NN-, -NH-, -CO-, -S-S-.
A method according to claim 1 wherein the groups R₁, R₂, R₃, R₄, and R₅ are the same or different and are independently selected from the group consisting of -H, -CO₂H, -OH, -Cl, -Br, - NH₂ or C_1-6 alkyl optionally substituted by a group -SO₃H, -CO₂H, - OPO₃H₂ or -OH, and, R₆ is selected from the group consisting of - O-, -OPO₃H₂, -CO₂H, -CONH₂, -CHO or C_1-6alkyl optionally substituted by a group -SO₃H, -CO₂H, -OPO₃H₂ or -OH.
A method according to claim 1, wherein the groups R₁, R₂, R₃, R₄, R₅ are hydrogen and the group R₆ is as defined in claim 1.
A method according to claim 1, wherein the groups R₁, R₂, R₄, and R₅ are hydrogen, the group R₃ is a C_1-6, alkyl optionally substituted by a group -SO₃H, -CO₂H or -OH and the group R₆ is as defined in claim 1.
A method according to claim 1, wherein the groups R₁, R₂, R₄, and R₅ are hydrogen, the group R₃ is an unbranched C_1-4 alkyl optionally at its terminal carbon by a group -SO₃H, -CO₂H or -OH and the group R₆ is as defined in claim 1.
The method according to any one of claims 2 to 5 wherein R₆ is a linear or branched C_1-6 alkyl group optionally substituted by a group -SO₃H, -CO₂H, -OH; oxygen; or a 4-pyridyl group or acid addition salt thereof.
The method according to claim 6 wherein R₆ is a methyl, ethyl, n-propyl, n-butyl, n-amyl or n-hexyl group optionally substituted by a group -SO₃ ^-, -CO₂ ^- or -OH.
The method of claim 6 wherein R₁, R₂, R₃, R₄, R₅ are hydrogen and R₆ is sulfomethyl, sulfoethyl, sulfopropyl or sulfobutyl.
The method according to claim 1 wherein said compound is:
(a) pyridine-N-oxide;

(b) 3-(1-pyridinio)-1-propanesulfonate; or

(c) 2-pyridinol 1-oxide.
The method according to any one of the preceding claims wherein said DNA polymerase reaction is:
(a) a PCR;

(b) a reaction of chain termination DNA sequencing;

(c) a primer extension reaction; or

(d) a nick-translation reaction.
The method according to any one of the preceding claims wherein the reaction mixture comprises a DNA molecule having trinucleotide repeats where the trinucleotides are GC rich.
A kit for performing a DNA-dependent DNA polymerase reaction comprising in separate containers:
a) components for DNA-dependent DNA polymerase reaction; and

b) a container which contains one or more compounds of the formula I:
wherein:
R₁, R₂, R₃, R₄, R₅ may be the same or different and are independently selected from the group consisting of -H, -OH, -F, -Cl, -Br, -CF₃, -CCl₃, -CBr₃, -NH₂, -NO₂, -CN, -CONH₂, -CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂ and a group R₇,

R₆ is selected from the group consisting of -O-, -OPO₃H₂, - CF₃, -CCl₃, -CBr₃, -CN, -CONH₂, -CHO, -CO₂H, and a group R₇,

each group R₇ is independently selected from C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or an aryl or heteroaryl ring containing from 5 to 8 ring members, which in the case of heteroaryl rings may include 1 or 2 nitrogen, oxygen or sulphur atoms or mixtures thereof, the remainder being carbon, and wherein R₇ is optionally be substituted by from 1 to 4 groups selected from - OH, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -NH-NH₂, , -NH₂, -NO, - NO₂, -CN, -CONH₂, -CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂, or mixtures thereof, and further optionally R₇ may comprise a group -NH-NH-, -NN-, -NH-, -CO-,
-S-S-.
The kit of claim 12 wherein the compound of formula I is as defined in any one of claims 2 to 9.
The kit of claim 12 or 19 further comprising a DNA-dependent DNA polymerase enzyme.
The kit of claim 14 wherein said DNA-dependent DNA polymerase enzyme is: (a) selected from the group consisting of E. coli DNA Polymerase I, Klenow enzyme, T4 DNA Polymerase, and T7 DNA Polymerase; or
(b) a thermostable DNA-dependent DNA polymerase.
The kit of claim 15 part (b) wherein said thermostable DNA-dependent DNA polymerase is selected from the group consisting of Taq DNA polymerase; N-terminal deletions of Taq polymerase, Tth DNA polymerase, Bacillus caldotenax DNA polymerase; Thermus flavus DNA polymerase; Bacillus stearothermophilus DNA polymerase; and archaebacterial DNA polymerases, such as Pfu DNA polymerase, Pfx DNA polymerase, Pwo DNA polymerase, Thermococcus litoralis DNA polymerase, and DeepVent_R ^®.
In a method of performing a DNA-dependent DNA polymerase reaction, the improvement comprising including in a reaction mixture containing a DNA-dependent DNA polymerase a compound of the formula I:
wherein:
R₁, R₂, R₃, R₄, R₅ may be the same or different and are independently selected from the group consisting of -H, -OH, -F, -Cl, -Br, -CF₃, -CCl₃, -CBr₃, -NH₂, -NO₂, -CN, -CONH₂, -CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂ and a group R₇,

R₆ is selected from the group consisting of -O-, -OPO₃H₂, - CF₃, -CCl₃, -CBr₃, -CN, -CONH₂, -CHO, -CO₂H, and a group R₇,

each group R₇ is independently selected from C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or an aryl or heteroaryl ring containing from 5 to 8 ring members, which in the case of heteroaryl rings may include 1 or 2 nitrogen, oxygen or sulphur atoms or mixtures thereof, the remainder being carbon, and wherein R₇ is optionally be substituted by from 1 to 4 groups selected from - OH, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -NH-NH₂, , -NH₂, -NO, - NO₂, -CN, -CONH₂, -CHO, -CO₂H, -SH, -SO₃H, -OPO₃H₂, or mixtures thereof, and further optionally R₇ may comprise a group -NH-NH-, -NN-, -NH-, -CO-, -S-S-.